Eicosapentaenoic acid (“EPA”; cis-5,8,11,14,17-eicosapentaenoic acid; ω-3) is an important intermediate in the biosynthesis of biologically active prostaglandin. Additionally, EPA is recognized as having clinical and pharmaceutical value. For example, the following pharmacological actions of EPA are known: 1) platelet coagulation inhibitory action (thrombolytic action); 2) blood neutral fat-lowering action; 3) actions for lowering blood VLDL-cholesterol and LDL-cholesterol and increasing HDL-cholesterol (anti-arterial sclerosis action); 4) blood viscosity-lowering action; 5) blood pressure lowering action; 6) anti-inflammatory action; and, 7) anti-tumor action. As such, EPA provides a natural approach to lower blood cholesterol and triglycerides. Increased intake of EPA has been shown to be beneficial or have a positive effect in coronary heart disease, high blood pressure, inflammatory disorders (e.g., rheumatoid arthritis), lung and kidney diseases, Type II diabetes, obesity, ulcerative colitis, Crohn's disease, anorexia nervosa, burns, osteoarthritis, osteoporosis, attention deficit/hyperactivity disorder, and early stages of colorectal cancer. See, for example, the review of McColl, J., NutraCos, 2(4):35-40 (2003); Sinclair, A., et al. In Healthful Lipids; C. C. Akoh and O.-M. Lai, Eds; AOCS: Champaign, Ill., 2005; Chapter 16. Recent findings have also confirmed the use of EPA in the treatment of mental disorders, such as schizophrenia (U.S. Pat. No. 6,331,568; U.S. Pat. No. 6,624,195). Lastly, EPA is also used in products relating to functional foods (nutraceuticals), infant nutrition, bulk nutrition, cosmetics and animal health.
Microbial production of EPA using recombinant means is expected to have several advantages over production from natural microbial sources (e.g., heterotrophic diatoms Cyclotella sp. and Nitzschia sp.; Pseudomonas, Alteromonas or Shewanella species; filamentous fungi of the genus Pythium; or Mortierella elongata, M. exigua, or M. hygrophila) or isolation from fish oil and marine plankton. For example, recombinant microbes having preferred characteristics for oil production can be used, since the naturally occurring microbial fatty acid profile of the host can be altered by the introduction of new biosynthetic pathways in the host and/or by the suppression of undesired pathways. This results in increased levels of production of desired polyunsaturated fatty acids [“PUFAs”], or conjugated forms thereof, and decreased production of undesired PUFAs. Secondly, recombinant microbes can provide PUFAs in particular forms which may have specific uses. Additionally, microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrate sources for microbially expressed enzymes, or by addition of compounds/genetic engineering to suppress undesired biochemical pathways. Thus, for example, it is possible to modify the ratio of ω-3 to ω-6 fatty acids so produced, or engineer production of a specific PUFA (e.g., EPA) without significant accumulation of other downstream or upstream PUFA products. Production of EPA in recombinant microbes also avoids use of non-sustainable oceanic sources, which can suffer from objectionable flavors and contaminants that are difficult and cost-prohibitive to remove. The resulting EPA oil isolated from fermentated recombinant microbes obviates the need for purification of these bioaccumulative compounds.
The literature reports a number of recent examples whereby various portions of the ω-3/ω-6 PUFA biosynthetic pathway, responsible for EPA production, have been introduced into plants (e.g., Qi, B. et al., Nature Biotech., 22:739-745 (2004)) and Saccharomyces cerevisiae (a non-oleaginous yeast) (see, Dyer, J. M. et al., Appl. Eniv. Microbiol., 59:224-230 (2002); U.S. Pat. No. 6,136,574; Domergue, F. et al., Eur. J. Biochem., 269:4105-4113 (2002)).
Yarrowia lipolytica has a number of characteristics that make it particularly useful for the production of PUFAs (see for example commonly owned U.S. Pat. No. 7,238,482). Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation is at least 25% of the cellular dry weight. Commercial production of EPA will require a strain that produces high amounts of EPA as a weight percent of the total fatty acids. Applicants have solved the stated problem by engineering highly optimized strains of Yarrowia lipolytica that are capable of producing greater than 53.2% EPA in the total oil fraction.